1. Technical Field
The present invention relates to a novel uracil-DNA glycosylase (hereinafter, “UDG”), a polynucleotide encoding the UDG, a recombinant vector comprising the polynucleotide, a host cell transformed by the vector, a method for producing the UDG, and a method for using the same.
2. Background Art
UDG has been known as an enzyme which repairs damaged DNA, by recognizing the damaged moiety of the DNA and hydrolyzing N-glycosylic bond between the deoxyribose sugar and the uracil base in the DNA so as to remove the damaged base from the DNA. UDG has been first isolated from E. coli, and then found in various bacteria including Bacillus. UDG has a molecular weight of about 25˜35 kDa and substrate specificity which specifically and selectively removes uracil bases, among other bases, from DNA [Refer to: Lindahl, T., Proc. Natl. Acad. Sci. USA 71, 3649-3653, 1974; Cone, R. et al., Biochemistry 16, 3194-3201, 1977].
Uracil is a base normally present in RNA, but sometimes found in DNA. Such presence of uracil in DNA may occur, when uracil generated by naturally-occurring deamination of cytosine is inserted into DNA, or when dUTP, instead of dTTP, is accidentally inserted into DNA during DNA replication process. With regard to this, UDG specifically removes uracil residues present in DNA, not uracil residues in RNA, thus forming an apyrimidinic (AP) site where a base is removed, and facilitating reactions of various DNA-repairing enzymes such as AP endonuclease, DNA polymerase, DNA ligase, or the like. Thereby, processes for repairing damaged or mutated DNA are carried out [See, Chen, R. et al., J Gen Virol. 83, 2339-2345, 2002; Lanes, O. et al., Extremophiles 6, 73-86, 2002].
Polymerase chain reaction (PCR) is a technique used for isolating or identifying useful genes by amplifying specific nucleic acid regions in large quantities in vitro, with the use of DNA polymerase originated from thermophiles and hyperthermophiles [See: Erlich, H. A., J Clin Immunol 9, 437-447, 1989; Shin, H. J. et al., J Microbiol Biotechnol 15, 1359-136, 2005]. The PCR technique has contributed to a lowering of the nucleic acid detection limit in a significant way, owing to its increased sensitivity. Currently, this technique is very effectively used for the detection and identification of diseases by detecting viruses and pathogens. However, when the concentration of a nucleic acid is very low, it is still difficult to detect the nucleic acid of interest. Further, it has a problem that the reaction efficiency is different depending on the reaction condition. Still further, one of the most significant problems of this technique in the use of clinical diagnosis is contamination of a sample, which may cause a wrong diagnosis such as false positive. Such contamination can further lead to cross contamination in the process of selecting samples, isolating nucleic acids, transferring the samples, PCR of samples, storing samples and collecting samples from electrophoresis. The sources of contamination during PCR may be cross contamination among samples, DNA contamination in a lab, and carry-over contamination between amplified products and primers of the previous PCR [See: Sobek, H. et al., FEBS Lett 388, 1996]. In the case of cross contamination among said contaminations, even if the degree of cross contamination is very small, it causes a problem in that contamination of a sample cannot be recognized with a conventional PCR technique, when it is amplified together with the sample of interest.
Therefore, in recent years, many methods for preventing cross contamination occurring after a PCR process have been developed. In one example of the methods, PCR is carried out by using dUTP instead of dTTP [See, Longo, M. C. et al., Gene 93, 125-128, 1990]. Another example of the methods comprises: adding a template DNA and UDG for removing a very small amount of contaminant, uracil-DNA in a sample; heating the mixture to inactivate UDG; adding thereto dUTP instead of dTTP; and carrying out PCR, have been reported. [See: Udaykumar., et al., Nucleic Acids Res. 21, 3917-3918, 1993; Taggart et al., J. Virol. Methods 105, 57-65, 2002]. In this respect, currently, PCR products which use UDG in the PCR process or contain UDG are commercially available.
However, UDGs originating from E. coli mesophiles are not completely inactivated at high temperature over 60° C., but maintain some of their residual activity so that the uracil-containing DNA product which has been amplified in PCR using dUTP undergoes degradation, resulting in reducing the amount of the final product. For example, in a Reverse Transcriptase-PCR (RT-PCR) using dUTP and mesophilic UDG, the first step of an RT-PCR process is conducted generally at a temperature in the range of 55° C. to 60° C. that is a maximum temperature range for the reaction of a reverse transcriptase, in order to unwind the secondary structure of RNA, and this results in significant decrease in the amount of PCR products, due to mesophilic UDG which maintains its residual activity. Therefore, a cumbersome step of inactivating UDG after UDG treatment for removing contaminated dUMP-containing DNA must be conducted, and then PCR with the use of dUTP is carried out.
Recently, development of psychrophilic UDG which is labile to heat, for making it possible to directly carry out PCR or RT-PCR without going through a UDG inactivating step after UDG treatment, has been receiving more attentions. However, there has been just one psychrophilic UDG which becomes easily inactivated by heat, reported so far, which is an enzyme isolated from a marine psychrophilic BMTU3346 [See: Jaeger, S. et al., Extremophiles 4, 115-122, 2000].
There is thus a need for a novel UDG which can solve the above-described problems associated with prior art.
The information disclosed in this Background of the Invention section is only for enhancement of understanding of the background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art that is already known to a person skilled in the art.